U.S. patent number 4,654,299 [Application Number 06/592,046] was granted by the patent office on 1987-03-31 for procedure for the irreversible binding of proteins onto polystyrene surfaces with retention of their biological activity, polystyrene surfaces obtained by this procedure, and their use.
This patent grant is currently assigned to Mallinckrodt Diagnostica (Germany) GmbH. Invention is credited to Dierck Lentfer.
United States Patent |
4,654,299 |
Lentfer |
March 31, 1987 |
Procedure for the irreversible binding of proteins onto polystyrene
surfaces with retention of their biological activity, polystyrene
surfaces obtained by this procedure, and their use
Abstract
A procedure for the immobilization of proteins on polystyrene
surfaces which includes a pre-treatment of the polystyrene surface
with a bis-diazonium compound of the general formula I where
##STR1## R1 stands for a hydrogen atom, a halogen atom, an alkyl
group, an alkoxy group or a nitro group and where R2 stands for a
hydrogen atom, a halogen atom or an alkyl group and where X stands
for an anion and the subsequent adsorption of the protein on the
surface pretreated. Proteins immobilized by its procedure may be
used in immunoassays.
Inventors: |
Lentfer; Dierck (Rodgau,
DE) |
Assignee: |
Mallinckrodt Diagnostica (Germany)
GmbH (Dietzenbach-Steinberg, DE)
|
Family
ID: |
6195287 |
Appl.
No.: |
06/592,046 |
Filed: |
March 21, 1984 |
Foreign Application Priority Data
|
|
|
|
|
Mar 31, 1983 [DE] |
|
|
3311889 |
|
Current U.S.
Class: |
435/7.92;
436/532; 436/800; 436/810; 436/531; 436/804 |
Current CPC
Class: |
G01N
33/545 (20130101); Y10S 436/81 (20130101); Y10S
436/804 (20130101); Y10S 436/80 (20130101) |
Current International
Class: |
G01N
33/544 (20060101); G01N 33/545 (20060101); G01N
033/53 (); G01N 033/545 (); G01N 033/549 () |
Field of
Search: |
;436/531-532,810,800,804
;435/7 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Nucker; Christine M.
Attorney, Agent or Firm: Klostermann; R. J. Goodwin; L. N.
Jackson; R. G.
Claims
What is claimed is:
1. Procedure for the irreversible binding of proteins onto a
polystyrene surface, comprising
(a) treating a polystyrene surface with a bis-diazonium compound
under bis-diazonium compound treating conditions of the general
formula I, ##STR4## where R1 stands for a hydrogen atom, a halogen
atom, an alkyl group, an alkoxy group or a nitro group and
where
R2 stands for a hydrogen atom, a halogen atom, an alkyl group and
where X stands for an anion and
(b) by thereafter adsorbing a protein on to the surface under
protein adsorbing conditions.
2. Procedure according to claim 1, where each R1 is ortho to the
nitrogen group and stands for a hydrogen atom, a methyl group or a
methoxy group and where each R2 is ortho to the nitrogen group and
stands for a hydrogen atom, a methyl group, and where X stands for
a halogen or tetrafluoroborate ion.
3. Procedure according to claim 2, where R1 a stands for a methoxy
group, R2 a stands for a hydrogen atom and X stands for Cl.sup.- or
BF.sub.4.sup.-.
4. Procedure according to claim 1, where the bis-diazonium compound
is in the form of a complex.
5. Procedure according to claim 1, wherein the protein is an
antibody, an antigen, a hapten protein conjugate, an
antibody-binding protein, an enzyme or lectin.
6. A bis-diazonium treated polystyrene vessel or bead suitable for
adsorbing proteins prepared by step (a) of claim 1.
7. A bis-diazonium treated polystyrene vessel or bead having
proteins adsorbed thereto prepared according to the procedure of
claim 1.
8. Polystyrene articles according to claim 7, wherein the protein
is an antibody, antigen, hapten protein conjugate, antibody-binding
protein, enzyme or lectin.
9. In an immunochemical or enzymatical method for determining the
concentration of an analyte in a measured amount of an aqueous
sample wherein said aqueous solution is contacted with:
(1) an insoluble carrier to which has been attached a biologically
active substance capable of reacting with said analyte and
(2) a measured amount of a tracer labeled member to form after
substantial equilibration a two-phase system containing a solid
phase having a portion of the labeled member and unlabeled member
bound to said biologically active substance and a liquid phase
containing the balance of the unbound labeled member and unlabeled
member,
(3) the two phases separated and the concentration determined,
(4) the improvement comprising using as the insoluble carrier the
article prepared by the procedure of claim 1.
10. A method according to claim 9 wherein the tracer label is
selected from the group consisting of a radio label, an enzyme
label, a fluorescent label, and a luminescent label.
Description
The invention concerns a procedure for the irreversible binding of
proteins onto polystyrene surfaces, polystyrene surfaces obtained
by this procedure and their use.
The immobilization of biological active proteins, such as enzymes,
antibodies, antigens, finds broad application for different
purposes, e.g. in enzyme reactors, for affinity chromatography, for
immune adsorption, for ligand assays with solid-phase
separation.
A special application is that of immobilized antigens and
antibodies in the field of immunoassays (e.g. RIA, ELISA, etc.) for
the phase separation in heterogeneous systems. The heterogeneous
immunoassay systems include one or more steps, by whch a separation
between bound and unbound portions of an analyte or reagent is
achieved. The binding consists in antigen-antibody
interactions.
The necessary separation can be performed elegantly, if the given
binding partner (the specific antibody or the specific antigen) is
irreversibly immobilized in reactive form on a smooth macroscopic
surface. The immobilization may be performed for example, on the
inner wall of the reaction vessel or on a bead which is covered by
the reaction liquid.
A smooth macroscopic surface is preferred to avoid a mechanical
separation by for example, centifugation or filtration as necessary
by microscopically distributed surfaces (e.g. latex) and to
decrease the amount of adhering reaction liquid (which is high with
porous materials as for example gels).
Polystyrene surfaces coated adsorptively with proteins have found
broad application in solid-phase separation immunoassay systems.
Polystyrene offers the following advantages:
low price
clear, transparent
reaction vessels of various kinds and beads commercially
available.
The main advantage of adsorptive coating is its simplicity: a
protein solution of appropriate concentration is brought into
contact with the plastic surface under protein adsorbing conditions
for a certain time, for example, 3-16 hours, thereafter the protein
solution is removed and the surface is washed. The yield is up to
60% of protein immobilized, the density is in the orger of 1 ug of
protein per cm.sup.2. The advantage of simple coating is contrasted
by drawbacks in the use: with weaker binding proteins a sufficient
density of coating is only achieved with a considerable excess of
protein; immobilization is reversible, under assay conditions (i.e.
in presence of other proteins or of Tween 20 for the prevention of
unwanted subsequent adsorption) or a loss of protein immobilized
(bleeding out) is observed, which may interfere with the assay.
Attempts exist to overcome these disadvantages of the adsorptive
immobilization by introduction of a covalent binding to the plastic
surface. Various procedures have been described, which make use of
glutarialdehyde as a coupling reagent. The mechanism involved has
not been identified, especially whether the improvements observed
are due to an involvement of the glutardialdehyde in the connection
to the plastic material, or if they are caused by a stabilization
of the biologically active conformation of the protein by
intramolecular bridging.
In addition, y-irradiation (cobalt-sterilization) of polystyrene
articles is used for the improvement of the adsorptive binding of
proteins, but this increases considerably the prices of
commercially available articles.
The objective of the present invention is therefore to make
available a simple and low-price procedure for the irreversible
binding of proteins onto polystyrene surfaces with retention of
their biological activity.
This objective is achieved:
(a) by the pre-treatment of the polystryene surface with a
bis-diazonium compound of the general formula I ##STR2## where R1
stands for a hydrogen atom, a halogen atom, an alkyl group, an
alkoxy group or a nitro group and where
R2 stands for a hydrogen atom, a halogen atom, an alkyl group and
where X stands for a halogen ion or a tetrafluoroborate-ion and
(b) by the subsequent, adsorption of the protein to the surface
treated in this way.
Alkyl or alkoxy groups are those with 1 to 4 carbon atoms,
preferably methyl and methoxy. Halogen atoms are bromine, chlorine
and fluorine, preferably chlorine. Preferred bis-diazonium
compounds are those of the general formula I a ##STR3## where R1a
stands for a hydrogen atom, a methyl group, or a methoxy group and
where
R2a stands for a hydrogen atom or a methyl group and where
X stands for a halogen ion or tetrafluoroborate-ion.
A preferred compound of formula Ia is that, where R1a stands for a
methoxy group, R2a stands for a hydrogen atom and X for Cl.sup.- or
BF.sub.4.sup.-.
Alternatively the bis-diazonium compounds I or Ia may be used in
other forms commercially available, e.g. as complexes. The
polystyrene surfaces may exist in any given form; e.g. as reaction
vessels, as reagent tubes, beakers, cuvettes, columns, microtiter
or microtest plates, or as parts, e.g. beads, rods, discs or
plates. Such polystyrene surfaces are well known to those skilled
in the art and are readily available.
The pre-treatment (activation) is performed under pre-treatment
conditions, e.g., at temperatures from about -5.degree. to about
+30.degree. C., preferably 4.degree.-10.degree. C. and activation
time is from about 5 to 60 min. The concentration of the
bis-diazonium compound is typically between about 10.sup.-5 and
10.sup.-1 moles per liter, preferably in a buffer of pH 6-8.
Suitable salts like NaCl, KI or NaIO.sub.4 may be added for
stabilization in stabilizing amounts.
Before the subsequent adsorption of protein, the polystyrene
surfaces are cleaned from unreacted bis-diazonium compound I by
washing with buffer and/or water. The adsorption may be performed
immediately after activation or with a time lag.
The subsequent adsorption of the protein is performed under protein
adsorbing conditions by incubation of the protein, dissolved e.g.
at a concentration from about 10.sup.-9 to 10.sup.-3 g/ml,
preferably 10.sup.-6 to 10.sup.-4 g/ml in a buffer of pH 6-8, for
about 1 to about 72 hours. Any protein may be used, e.g.
antibodies, antigens, hapten protein conjugates, antibody-binding
proteins, for example, staphylococcal protein A or complement
component C.sub.1q, enzymes, lectins.
The polystyrene surface with the absorbed (immobilized) protein may
be used for the desired purposes immediately after the adsorption
or after storage.
The activation of the polystyrene surfaces leads to a practically
irreversible binding of proteins, which enables an increase in
sensitivity, and decrease in material consumption in e.g. immuno
assays (no bleeding out). The activation leads to a stabilization
of the adsorbed proteins, especially when a subsequent drying step
is performed.
The polystyrene articles may be distributed commercially after the
activation or after the protein adsorption or in the form of a kit.
If distributed in the form of a kit, this includes besides the
polystyrene article a test tube or a microtest plate, other usual
components, for example, a labelled antibody and a detecting
reagent for the marker, e.g. a chromogenic substrate.
The bis-diazonium compounds I are well known and are prepared from
the corresponding benzidines according to well-known procedures.
(Houben-Weyl, Methoden der organischen Chemie, Georg Thieme Verlag
Stuttgart, Vol. 10/3, pp. 1-212, esp. 46; Ullmanns Enzyklopadie der
technischen Chemie, 4. Auflage Verlag Chemie Weinheim, Vol. 8, p.
356 ff.)
The following examples serve for the illustration of the invention,
without restricting it. Temperatures are given in .degree.C.
GENERAL WORKING PROCEDURE
1. The polystyrene surfaces are completely covered for 30 minutes
at 40.degree. with a solution of 0.001 moles per liter of
FBS(BF.sub.4).sub.2 (FBS--Fast Blue B Salt=bis-diazotized
o-dianisidine) in 0.01 moles per liter sodium phosphate buffer pH
6.8, under protection from direct light. The FBS solution is
removed, the polystyrene articles are washed 3 times with 0.01 mole
per liter sodium phosphate buffer.
2. Protein Adsorption
The polystyrene articles pre-treated according to 1. are incubated
with a solution or suspension of the desired protein (maximal 2 ug
of protein per cm.sup.2 of surface to be coated) in 0.01 moles per
liter sodium phosphate buffer pH 6.8 for 16 hours at 4.degree.. The
protein solution is removed, weakly bound protein is removed by
incubation with sodium phosphate buffer containing 0.1%
Tween.RTM.20 (Polysorbate 20=Polyoxyethylene 20 sorbitan
monolaurate) for 1 hour.
SPECIFIC EXAMPLES
1. .sup.125 I-labled, y-globulin (from goat, 140000 cpm/ug protein)
was coated according to A.2. onto untreated polystyrene tubes
(11.times.65, Greiner, Germany) and onto the same tubes pre-treated
according to A.1. The volumes of the FBS and y-globulin solutions
were 1 ml each, the y-globulin solution was applied in a range of
graduated concentrations: 1; 0.5; 0.25; 0.125; and 0.0625 and
0.03125 ug/ml.
Table 1 shows the results obtained:
TABLE 1 ______________________________________ Coating yield (in %)
.sup.125 I-Y-globulin without FBS with FBS applied pre-treatment
pre-treatment (ug) I II I II ______________________________________
1 64.8 24.9 57.2 54.4 0.5 69.1 20.3 62.6 59.6 0.25 66.2 18.5 60.6
57.1 0.125 68.8 18.7 62.8 60.5 0.0625 65.7 17.3 62.4 59.2 0103125
59.5 14.9 67.6 63.3 ______________________________________ I:
before treatment with Tween II: after treatment with Tween
The coating yield without FBS-pre-treatment was about 65%, with FBS
pre-treatment it was about 60%. If both kinds of coated tube were
incubated for 1 hour with a 0.1% solution of Tween 20 in
physiological saline, the coating yield of the tubes without FBS
pre-treatment dropped to about 20%, whereas it remained practically
unchanged with the pre-treated tubes.
2. According to B.1. untreated and FBS pre-treated polystyrene
tubes were ocated with Theophylline-antiserum (from rabbit, 1 ml
per tube, dilution 1:36000). The tubes were subsequently washed for
1 hour with a 1% solution of polyvinyl alcohol (low molecular
weight type) in 0.01 mole per liter potassium phosphate buffer pH
7.4 The coated tubes were incubated for 1 hour at room temperature
with 1 ml each of a solution of Theophylline-peroxidase conjugate
(10.sup.-7 g/ml, about 2 molecules of Theophylline per molecule of
horse radish peroxidase) in 0.01 moles per liter potassium
phosphate buffer pH 7.4 containing 0.1% gelatine, 0.9% NaCl and
0.03% magnesium-1-anilinonaphthalene-8-sulfonate. Subsequently the
tubes were washed with cold tap water (3 times). The determination
of antibody-bound enzyme activity was achieved by incubating the
tubes for 20 minutes at room temperature with 1 ml of a solution
containing 0.33 mg/ml o-phenylenediamine and 0.05 mg/ml H.sub.2
O.sub.2 in 0.1 mol per liter tris-acetate buffer pH 5.6. The
reaction was stopped by addition of 1 ml of 1 mole per liter
sulfuric acid. The optical density at 492 was determined
photometrically. The result was 0.129 for untreated and 0.944 for
FBS pre-treated tubes.
3(a) According to B.1. untreated and FBS pre-treated polystyrene
microtiter plates were coated with 0.1 ml/cup of various dilution
sof a streptolysin-o-preparation (0.131 mg/ml protein; about 4000
IU/mg protein).
1:2 dilution series (volume 0.1 ml/cup) of an anti-streptolysin
standard serum (Behring-Werke, Marburg; 10 IU/ml) were prepared in
the differently coated microtiter plates, using 0.1 mole per liter
Tris-HCl buffer pH 7.8 with 0.1% Tween 20 as the diluent. The
plates were incubated for 30 minutes at 37.degree. C., the serum
dilutions were removed and the plates were washed once with cold
tap water. To each cup 0.1 ml of a solution containing 2 ug/ml of
an anti-Hu IgG-peroxidase conjugate (rabbit IgG against human IgG,
coupled to horse radish peroxidase) was added and incubated for 30
minutes at 37.degree. C. After removal of the conjugate solution
the plates were washed 3 times with cold tap water. For the
detection of peroxidase activity immobilized by immuno adsorption,
0.1 ml per cup of a substrate mixture (0.16 mg/ml o-tolidine, 0.05
mg/ml H.sub.2 O.sub.2 in 0.1 mole per liter tris-citrate buffer pH
5) was added and incubated for 30 minutes at room temperature. For
evaluation the highest serum dilution still producing a blue color
was identified.
The results are shown in Table 2.
TABLE 2 ______________________________________ Streptolysin Highest
positive dilution of dilution standard serum used for coating
without FBS with FBS ______________________________________ 1:20 40
80 1:40 20 80 1:80 10 20 ______________________________________
3(b) The differences are even more pronounced, if the coating
procedure is followed by a drying step (3 hours at room temperature
in vacuo at 0.1 mbar), which is advantageous for longer
storage.
Table 3 shows the results obtained according to 3(a) (Streptolysine
dilution 1:80; highest positive dilutions given for standard serum
and the patient sera). The better performance of the activated
plates can be seen.
TABLE 3 ______________________________________ Highest Positive
Dilution of Serum Sample without FBS with FBS
______________________________________ Standard serum negative 60
Patient I 200 6400 Patient II 150 6400 Patient III 50 4800
______________________________________
4. According to B.1. untreated or FBS-treated polystyrene
microtiter plates were coated with 0.1 ml per cup of a solution
containing 0.01 mg/ml of a glycoprotein fraction from Candida
albicans (preprepared by Dr. H. Mauch, University des Saarlandes,
Homburg/Saar, Germany).
1:4600 dilutions of the serum samples given below in 0.1 mole per
liter Tris-HCl buffer pH 7.8 with 0.1% Tween.RTM. 20 were prepared
and were incubated for 1 hour at room temperature with 0.1 ml per
cup in the coated plates.
Serum A: healthy proband with low antibody concentration against
Candida albicans.
Serum B: pooled sera from healthy donors.
Serum C: patients serum I with elevated antibody concentration
against Candida albicans.
serum D: patients serum II with elevated antibody concentration
against Candida albicans.
After removal of the serum samples the plates were washed once with
cold tap water. To each cup 0.1 ml of a solution containing 0.1
ug/ml of an anti-Hu IgG-peroxidase conjugate (rabbit IgG against
human IgG coupled to horse radish peroxidase) was added and
incubated for 1 hour at room temperature. After removal of the
conjugate solution the plates were washed 3 times with cold tap
water. For the detection of peroxidase activity immobilized by
immunoadsorption 0.1 ml per cup of a substrate mixture (0.1 mg/ml
3,3',5,5'-Tetra methylbenzidine, 0.06 mg/ml H.sub.2 O.sub.2 in 0.1
mole per liter tris-citrate buffer pH 5) was added and incubated
for 30 minutes at room temperature. The reaction was stopped by
addition of 0.1 ml per cup of 1 mole per liter sulfuric acid and
the adsorption at 455 nm was determined photometrically in a
Kontron SLT 210 micro-titer plate reader.
The results are shown in table 4.
TABLE 4 ______________________________________ Absorption at 455 nm
Serum Sample without FBS with FBS
______________________________________ A 0.078 0.177 B 0.154 0.566
C 0.308 1.088 D 0.529 1.662
______________________________________
5. Kit for the determination of antibodies against streptolysine-o
in human serum.
The kit consists of:
(a) a micro-titer plate coated with Streptolysine-o according to
the invention described, serving as an antigen-coated reaction
vessel
(b) a solution of conjugate of rabbit IgG and horse radish
peroxidase, directed against human IgG (lyophilized, if
necessary)
(c) 0.1 mole per liter tris citrate buffer pH 5 for the peroxidase
reaction
(d) a stock solution of 3,3',5,5'-tetramethylbenzidine in
dimethylsulfoxide (0.1 mg/ml), serving as a chromogen for the
peroxidase reaction
(e) a solution of hydrogen peroxide (3%) for the peroxidase
reaction
(f) a 0.1 mole per liter tris-HCl buffer solution pH 7.8,
containing 0.1% Tween.RTM. 20, serving as a diluent for serum
samples and antibody-peroxidase conjugate.
* * * * *